Unnatural amino acids can be site-specifically incorporated into polypeptides with high efficiency and high fidelity by means of heterologous orthogonal tRNA/aminoacyl-tRNA synthetase pairs (O-tRNA/O-RS pairs) (Deiters, et al. (2003) “Adding Amino Acids with Novel Reactivity to the Genetic Code of Saccharomyces cerevisiae.” J Am Chem Soc 125: 11782-11783; Wang, et al. (2001) “Expanding the Genetic code of Escherichia coli.” Science 292: 498-500; Chin, et al. (2003) “An Expanded Eukaryotic Genetic Code.” Science 301: 964-7). These O-tRNA/O-RS pairs recognize their cognate unnatural amino acids but do not significantly cross-react with the tRNAs, aminoacyl tRNA synthetases or amino acids that are endogenous to the system in which they are being used. To date, this technology has permitted the genetically encoded incorporation of more than 30 different unnatural amino acids with unique steric and/or chemical properties into proteins synthesized in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells (Xie, J, et al. (2006) “A chemical toolkit for proteins—an expanded genetic code.” Nature Rev Mol Cell Biol 7:775-782; Wang, L, et al. (2005) “Expanding the genetic code.” Agnew Chem Int Edit 44: 34-66, Liu, et al. (2007) “Genetic incorporation of unnatural amino acids into proteins in mammalian cells.” Nature Methods 4: 239-244). This methodology can be particularly useful in the development and large-scale production of therapeutic proteins with enhanced biological properties, reduced toxicities, and/or increased half-lives.
E. coli and S. cerevisiae expression systems are widely used to synthesize heterologous proteins and can be adapted for large-scale synthesis of proteins comprising unnatural amino acids (Adding Amino Acids with Novel Reactivity to the Genetic Code of Saccharomyces Cerevisiae.” J Am Chem Soc 125: 11782-11783; Wang, et al. (2001) “Expanding the Genetic code of Escherichia coli.” Science 292: 498-500). However, neither of these expression systems is well suited for the production of recombinant mammalian proteins, which often require sulfation, glycosylation or post-translational modifications in order to exhibit a desired biological activity. Furthermore, neither of these hosts is optimal for the production of therapeutic proteins: Proteins produced in E. coli usually contain high concentrations of pyrogenic compounds, e.g., endotoxin, and proteins synthesized in S. cerevisiae can contain potentially antigenic α1,3 glycan linkages.
In contrast, methylotrophic yeast, such as Pichia pastoris, have been identified as attractive candidates for use as recombinant expression systems for heterologous proteins (Lin-Cereghino, et al. (2000) “Heterologous protein expression in the methylotrophic yeast Pichia pastoris.” FEMS Microbiol Rev 24: 45-66). The eukaryotic subcellular organization of methylotrophic yeast enables them to carry out many of the posttranslational folding, processing and modification events required to synthesize biologically active mammalian proteins. Unlike proteins expressed in S. cerevisiae, proteins produced by methylotrophic yeast such as P. pastoris are less likely to contain high-mannose glycan structures that can hamper downstream processing of heterologously expressed glycoproteins. In addition, proteins synthesized in methylotrophic yeast are free of pyrogenic and antigenic compounds.
Methylotrophic yeast expression systems are particularly useful for large-scale protein synthesis. For example, the yeast P. pastoris enables expression of recombinant proteins at levels 10- to 100-fold higher than in S. cerevisiae, bacterial, insect, or mammalian systems. In addition, methylotrophs such as P. pastoris can be easily cultured in a simple, defined salt medium, eliminating the need for the expensive media supplements and equipment that are required for baculovirus expression systems or mammalian tissue culture. Furthermore, P. pastoris is amenable to genetic manipulation, and many molecular microbiological techniques that have been developed for use with S. cerevisiae can be adapted for use in P. pastoris. 
What is needed in the art are new strategies for the site-specific incorporation of unnatural amino acids into proteins in a low-cost expression system that is capable of producing biologically active heterologous proteins that comprise complex posttranslational modifications. There is a need in the art for the development of O-tRNA/O-RS pairs and expression systems that function to incorporate unnatural amino acids into polypeptides synthesized in methylotrophic yeast. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.